Nuclei isolated from the livers of mice are capable of binding [125I]insulin. A class of high-affinity binding sites having a Kd of 1--2 nM and a capacity of approximately 2000 insulin molecules/nucleus are present on these nuclei. Removal of nuclear membranes by Triton X-100 treatment of the nuclei reduces or eliminates the high-affinity binding sites. Nuclei prepared from livers of the genetically obese mouse (ob/ob) lack, or have markedly reduced numbers of, the high-affinity binding sites whether or not the obese nuclei have been exposed to Triton X-100. The reduced insulin-binding capacity of the obese nuclei correlates with the reported decreased binding of insulin to plasma membranes prepared from target tissue of these animals. The possible physiological significance of nuclear insulin binding is discussed.